DESCRIPTION (from applicant's abstract): The addictive properties of narcotic analgesices result in widespread drug abuse and are undesirable side-effects of their use in the treatment of pain. Despite the importance of opioid drugs, our knowledge of the mechanisms underlying their addictive properties remains unclear. Activation of the mu opioid receptor is a necessary first step in producting opioid analgesia and other acute pharmacological effects, but it also appears to initiate a series of more poorly defined chronic changes associated with addiction. In this project we will address the following questions: What signaling pathways of the mu receptor are responsible for the acute and chronic effects of narcotic analgesics? Are these pathways distinct, and which ones are involved in regulating tolerance and dependence, two of the main questions in the following way: "First: we propose that mu receptors have basal activity--signaling activity in the absence of agonist--and that this basal activity increases after prolonged agonist treatment. Increased basal activity may represent a sensitization process. In contrast, agonist-dependent stimulation of mu receptor signaling is known to desensitize. Both the enhanced basal activity and the blunted agonist response could contribute to tolerance and dependence. We propose that differential phosphorylation of the mu receptor plays a role in this dual regulation, leading to both sensitization and desensitization. Second, based on recently obtained evidence we propose that calmodulin binds directly to the third intracellular loop pf mu opioid receptors. This is the first example of a direct interaction of calmodulin with a G protein coupled receptor. We will study the possible role of Ca2+ and calmodulin in reguling G protein coupling and phosphorylation of the mu opioid receptor. On the other hand, calmodulin could also serve as an alternate second messenger of mu receptor signaling per se, contributing to chronic adaptations associated with addiction. Third, we propose that the mu receptor exists in multiple conformation or functional states. This may result in varying ligand binding affinities and second messenger coupling. In addition to G protein coupling, we will examine activation of Ca++ influx and calmodulin as alternative signaling pathways of the mu opioid receptor. To test this hypothesis, we will screen a panel of opioid drugs and peptides for ability to stimulate these multiple signaling pathways. Further, we will test the hypothesis that the observed differential agonist effects could result from receptor aggregation into signaling complexes with effector proteins, such as calcium channels. Considered together, these hypotheses may yield important insights into the mechanisms underlying tolerance and dependence, providing the opportunity to develop effective therapies of narcotic addiction.